Molecular Epidemiology Of Porcine Kobuvirus And Its Prevention And Control Technology

PKV belongs to the family picornaviridae, genus Kobuvirus.Three species of viruses in the genus Kobuvirus are now known, including Aichi virus, Bovine kobuvirus and PKV. PKV was discovered in Hungary and China in 2007 and had since been detected and isolated in Thailand, Japan, Korea and Brazil, so it had circulated throughout the world.According to the reports, the positive rate of this virus in pigs with the symptom of diarrhea was significantly higher than those without it, but whether is it related with intestinal disease remains unclear. In order to understand the molecular epidemiology of PKV in Gansu Province, we conducted an epidemiological survey in the main hoggerys of this province. Overall, 126 samples were positive for PKV. Of the 74 diarrheic samples among the 203 fecal samples, 65 were positive for PKV. Because purified virus was the basis of studying the gene function, protein structure and physicochemical characteristics of the virus, but the isolation and purification of PKV are still not completed successfully, so we carried out this study with the reverse genetics technology. By analyzing the genome of CH441 and sequence of pBluscriptKS(Ⅱ) vector, we selected the appropriate enzyme digest sites, amplified the segments of the PKV genome and successfully constructed the full length plasimd of PKV CH441.In addition, as one of the most serious diseases that do harm to the breeding industry, viral diseases not only affect people’s life but also pose a serious threat to human health. How to control the occurrence and development of this disease has become a problem that we must overcome now. As a common lyase of the family picornaviridae, 3C protein plays very important role not only in the process of protein maturation but also in the aspect of antiviral drugs targeting. Therefore, the prokaryotic expression of 3C protein and analysis of its structure are important for the preparation of its crystals. Besides, as a nonstructural protein of PKV, 3C protein polyclonal antibody can be used to distinguish wild virus infection and vaccine virus infection. In this study, we analyzed the structure of PKV 3C gene, and obtained 3C protein by prokaryotic expression, and then we prepared the polyclonal antibody of 3C protein, which laying a strong foundation for the treatment and detection of PKV. In recent years, as an effective treatment of diseases, gene therapy is being widely accepted. In order to explore the method of gene therapy for PKV, we screened out and synthesized three sh RNA fragments against VP0, VP3 and 3A gene sequences respectively, and packaged them into lentiviral vectors and we obtained the stable transfected cell lines. It lays a theoretical basis for the study of functional genomics and new gene therapy drugs.